Fig. 6

TSG101 depletion decreases MMP-9 secretion and reduces cell invasion in PMA-treated HeLaS3 cells. Cells were transfected with control (con), TSG101 (TSG#1 or #2), or MMP-9 siRNA. a. Depletion of TSG101 by siRNA. Total cell lysates were analyzed by western blot. b–c. MMP-9 secretion and MMP-9 mRNA expression in TSG101-depleted cells. Cells were incubated in fresh serum-free medium containing or not 200 nM PMA for 24 h. MMP9 in conditioned media was measured using gelatin zymography (b, left). The amount of MMP-9 in PMA-treated cells was quantified and is expressed as arbitrary units (b, right). The amount of MMP-9 in conditioned media of the cells transfected with control (con) siRNA is set to 1.0. Expression levels of MMP-9 and GAPDH mRNAs in PMA-treated cells were analyzed by RT-PCR (c). The ratio of MMP-9 mRNA level relative to the GAPDH mRNA level is expressed as arbitrary units. The MMP-9 mRNA level in the cells transfected with control (con) siRNA is set to 1.0. d–e. Cell migration of TSG101-depleted cells. Confluent transfected cells were scratched and incubated in fresh serum-free medium containing 200 nM PMA for 9 h (d). Scale bars, 200 μm. Cell migration into the wound area in (d) was quantified (e). Relative migration activities of the cells transfected with TSG101 siRNA are expressed as the proportion of migration of the cells transfected with control (con) siRNA. f–g. Cell invasion of TSG101-depleted cells. Each of the transfected cells was allowed to invade for 72 h (f). Scale bars, 200 μm. Cell invasion through the filter in (f) was quantified (g). Relative invasion activities of the cells transfected with TSG101 or MMP-9 siRNA are expressed as the proportion of infiltration of the cells transfected with control (con) siRNA. The blots, gels, and images shown are representative of three independent experiments. The results shown are the means ± S.D. of three independent experiments. * p < 0.05; **, p < 0.005; ns, not significant, by a Student’s t-test